U.S. patent number 7,691,372 [Application Number 11/548,269] was granted by the patent office on 2010-04-06 for use of antibodies for the vaccination against cancer.
This patent grant is currently assigned to Igeneon Krebs-Immuntherapie Forschungs-und Entwickungs-AG. Invention is credited to Helmut Eckert, Hans Loibner.
United States Patent |
7,691,372 |
Eckert , et al. |
April 6, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Use of antibodies for the vaccination against cancer
Abstract
Described is the use of antibodies which are directed against
human cellular membrane antigens for the vaccination against cancer
diseases.
Inventors: |
Eckert; Helmut (Oberwil,
CH), Loibner; Hans (Vienna, AT) |
Assignee: |
Igeneon Krebs-Immuntherapie
Forschungs-und Entwickungs-AG (Vienna, AT)
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Family
ID: |
4178206 |
Appl.
No.: |
11/548,269 |
Filed: |
October 10, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070224202 A1 |
Sep 27, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09889300 |
Sep 13, 2001 |
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Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
A61P
35/00 (20180101); C07K 16/30 (20130101); C07K
16/28 (20130101); A61K 2039/55566 (20130101); A61K
2039/505 (20130101) |
Current International
Class: |
A61K
39/395 (20060101) |
Field of
Search: |
;424/130.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-96 01126 |
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Jan 1996 |
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WO |
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WO-97 15597 |
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May 1997 |
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WO |
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WO-98 56416 |
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Dec 1998 |
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WO |
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WO-98 56419 |
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Dec 1998 |
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WO |
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WO-99 65523 |
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Dec 1999 |
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WO |
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Primary Examiner: Yaen; Christopher H
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of application Ser. No.
09/889,300 filed on Sep. 13, 2001 now abandoned the entire contents
of which are hereby incorporated by reference and for which
priority is claimed under 35 U.S.C. .sctn.120.
Claims
The invention claimed is:
1. A pharmaceutical composition for treatment of cancer comprising
at least one antibody directed against the cellular membrane
antigen Ep-CAM and aluminum hydroxide, wherein said antibody
contains the idiotype of the HE2 antibody, and is contained in a
dosage range of 0.01 to 4 mg.
2. The pharmaceutical composition of claim 1, wherein said antibody
is of animal origin.
3. The pharmaceutical composition of claim 1, wherein said antibody
is a monoclonal antibody.
4. The pharmaceutical composition of claim 3, wherein said antibody
is a murine monoclonal antibody, wherein the variable region of the
heavy chain is the amino acid sequence as shown in SEQ ID NO: 1 and
wherein the variable region of the light chain is the amino acid
sequence as shown in SEQ ID NO: 2.
5. The pharmaceutical composition of any one of claims 1-3, wherein
said antibody has the same fine specificity of binding as the
antibody defined in claim 4.
6. The pharmaceutical composition of claim 1, wherein said
antibodies are directed against different epitopes of the membrane
antigen.
7. The pharmaceutical composition of claim 1, further comprising at
least one vaccine adjuvant.
8. A pharmaceutical composition for treatment of cancer disease
comprising a first antibody directed against the cellular membrane
antigen Ep-CAM and aluminum hydroxide, wherein said first antibody
is a murine monoclonal antibody, wherein the variable region of the
heavy chain is the amino acid sequence as shown in SEQ ID NO: 1 and
wherein the variable region of the light chain is the amino acid
sequence as shown in SEQ ID NO: 2 and is contained in a dosage
range of 0.01 to 4 mg.
9. The pharmaceutical composition of claim 8, wherein said antibody
is of animal origin.
10. The pharmaceutical composition of claim 8, wherein said
antibody is of monoclonal antibody.
11. The pharmaceutical composition of claim 8, further comprising
at least a second antibody directed against a different membrane
antigen or against a different epitope of said Ep-CAM membrane
antigen.
12. The pharmaceutical composition of claim 8, further comprising
at least one vaccine adjuvant.
13. The composition according to claims 1 or 8, further comprising
at least one adjuvant selected from the group consisting of
Bacillus Calmette Guerin, tetanus toxoid, Pseudomonas exotoxin,
Granulocyte Macrophage Stimulating Factor, interleukin-2, gamma
interferon, derivatives of lipopolysaccharides or combinations
thereof.
14. A method of treating individuals with cancer or identified as
being at risk of developing cancer comprising administering to a
patient in need thereof the pharmaceutical composition of claim
1.
15. The method according to claim 14, wherein said pharmaceutical
composition is administered by subcutaneous, intradermal or
intramuscular injection.
16. A method of treating cancer disease comprising administering to
a patient in need thereof the pharmaceutical composition of claim
8.
17. The method according to claim 16, wherein said pharmaceutical
composition is administered by subcutaneous, intradermal or
intramuscular injection.
18. The method according to claim 16, wherein said pharmaceutical
composition is administered by subcutaneous, intradermal or
intramuscular injection.
19. The method according to claims 16 or 17, wherein said dosage is
0.5 mg antibody.
Description
The present invention relates to the use of antibodies which are
directed against human cellular membrane antigens for the
preparation of a pharmaceutical composition for the vaccination
against cancer.
With the discovery of the hybridoma technology it became possible
to generate monoclonal antibodies (MAB) against the most varied
antigens. This technology which can generally be applied to all
biological problems also plays an important role in cancer
research. Over the last twenty years MAB directed against a
multitude of tumor-associated antigens (TAA) have been produced.
TAA are structures which are expressed predominantly on the cell
membrane of tumor cells and which, thus, allow differentiation from
non-malignant tissue. Therefore, they are regarded as targets for
diagnostic or therapeutic applications on the basis of specific MAB
or derivatives derived from these MAB.
Direct therapeutic applications of MAB which are directed against
TAA are based on passive immunotherapies, i.e. an MAB or a
derivative is applied systemically to cancer patients in a suitable
amount and has a therapeutic effect only as long as the
concentration in the organism is sufficiently high. The biological
half-life of such agents depends on their structure and ranges from
only a few hours to several days. It is therefore necessary to
repeat the applications. However, if xenogenic antibodies (e.g.
murine MAB) are used, this leads to unwanted immune reactions,
which can lead to the neutralization of a possible therapeutic
effect and to dangerous side effects (anaphylactic shock).
Therefore, such immunotherapeutics can only be administered for a
limited period of time.
Another approach for the immunotherapy of cancer is based on the
selective activation of the immune system of cancer patients so as
to combat malignant cells for which the most varied types of cancer
vaccines are used. These include vaccinations with autologous or
allogenic tumor cells, vaccinations with autologous or allogenic
tumor cells which have been chemically modified or which have been
modified by gene technological techniques, vaccinations with
isolated TAA or TAA which have been produced using chemical or gene
technological methods, with peptides derived therefrom, and,
recently, also vaccinations with DNAs coding for TAA or structures
derived therefrom, etc. An alternative method is based on the use
of anti-idiotypic antibodies for the vaccination against cancer.
Suitable anti-idiotypic antibodies can immunologically mimic a TAA.
As xenogeneic proteins (e.g. murine antibodies, goat antibodies
etc.) they induce a strong immune response in human after
vaccination--in contrast to the proper human tumor antigens, which,
as structures of the self, are often immunogenic to a low degree
only. Therefore, anti-idiotypic antibodies can be used for
vaccination as an immunogenic substitute for a tumor antigen.
In contrast to the passive immunotherapy with anti-tumor antibodies
in the active specific cancer immunotherapy, even very small
amounts of a suitable vaccine are, in principle, sufficient to
induce an immunity which lasts for months or for years and which
can be boosted by repeated vaccinations if it weakens. Moreover,
active immunization allows to induce a humoral as well as a
cellular immunity the cooperation of which can lead to an effective
protection.
In summary, the use of antibodies or their derivatives for
immunotherapy against cancer, which has been described so far, is
essentially based on two principles: passive therapy with
antibodies or their derivatives which are directed against TAA.
active immunization (vaccination) with antibodies or their
derivatives which are directed against the idiotype of antibodies
having a specificity against TAA.
In the course of the discovery and the subsequent characterization
of the most varied TAA, it has turned out that they have important
functions as regards cancer cells. They enable the degenerate cells
to show properties characteristic of the malignant phenotype, such
as an increased capability for adhesion, which play an important
role in establishing metastases. However, such antigens can, at
certain stages, also be expressed on normal cells where they are
responsible for the normal functions of these cells. Without laying
claim to completeness, some examples of such antigens are listed in
the following: N-CAM (Neuronal Cell Adhesion Molecule), which is
often expressed on tumors of neuronal origin and which effects
homophilic adhesion (J. Cell Biol. 118 (1992), 937). The Lewis Y
carbohydrate antigen, which occurs on the majority of tumors of
epithelial origin, but which also plays an important role during
the fetal development of epithelial tissues. It has been shown that
the expression of this antigen in lung cancer is strongly
associated with an unfavorable prognosis since Lewis Y positive
cancer cells obviously have a higher metastatic potential (N. Engl.
J. Med. 327 (1992), 14). CEA (Carcino Embryonic Antigen), which
often occurs on epithelial tumors of the gastrointestinal tract and
which has been identified as a self-adhesion molecule (Cell 57
(1989), 327). Ep-CAM (Epithelial Cell Adhesion Molecule), which is
expressed on nearly all tumors of epithelial origin, but which also
occurs on a large number of normal epithets. It has been
characterized as a self-adhesion molecule and can therefore be
classified as a pan-epithelial adhesion antigen (J. Cell Biol. 125
(1994), 437).
The technical problem underlying the present invention is to
provide further means and methods which allow an efficient
prophylaxis against or therapy of cancer diseases.
This problem has been solved by the provision of the embodiments as
characterized in the claims.
Accordingly, the invention relates to the use of antibodies which
are directed against human cellular membrane antigens for the
preparation of a pharmaceutical composition for the prophylactic
and/or therapeutic vaccination against cancer. In this context the
term "cellular membrane antigens" relates to structures which are
presented on the cell membrane of cells. These include in
particular receptors, such as the transferrin receptor, or other
molecules, such as E cadherine or Ep-CAM.
In a preferred embodiment, such a cellular membrane antigen is a
tumor-associated antigen. In this context, the term
"tumor-associated antigen" means a structure which is predominantly
presented by tumor cells and thereby allows a differentiation from
non-malignant tissue. Preferably, such a tumor-associated antigen
is located on or in the cell membrane of a tumor cell. This does,
however, not exclude the possibility that such antigens also occur
on non-degenerate cells. The tumor-associated antigens can, for
example, be polypeptides, in particular glycosylated proteins, or
glycosylation patterns of polypeptides. Other structures which may
represent a tumor-associated antigen are, e.g., glycolipids. These
include, for example, gangliosides, such as GM2. Moreover,
tumor-associated antigens may be represented by changes in the
composition of lipids of the cell membrane which may be
characteristic of cancer cells.
Examples of tumor-associated antigens are N-CAM, the Lewis Y
carbohydrate antigen, CEA and Ep-CAM, which have already been
mentioned above. Further examples are Sialyl Tn carbohydrate, Globo
H carbohydrate, gangliosides such as GD2/GD3/GM2, Prostate Specific
Antigen (PSA), CA 125, CA 19-9, CA 15-3, TAG-72, EGF receptor,
Her2/Neu receptor, p97, CD20 and CD21. Monoclonal antibodies
directed against all these antigens are available. Further
tumor-associated antigens are described, e.g., in DeVita et al.
(Eds., "Biological Therapy of Cancer", 2. Edition, Chapter 3:
Biology of Tumor Antigens, Lippincott Company, ISBN 0-397-51416-6
(1995)).
The term "antibody" relates to antibodies of all possible types, in
particular to polyclonal or monoclonal antibodies or also to
antibodies produced by chemical, biochemical or gene technological
methods. Methods for producing such molecules are known to the
person skilled in the art. The way of producing the antibody is not
important. Only its binding specificity for a relevant epitope of a
cellular membrane antigen is important. Preferably, monoclonal
antibodies are used, most preferably monoclonal antibodies of
animal origin, in particular of murine origin. It is particularly
preferred that the murine monoclonal antibody HE-2 is used, which
can be produced as described, or an antibody which has the same
fine specificity of binding as HE2.
Within the meaning of the present invention, the term "antibody"
also includes fragments and derivatives of antibodies wherein these
fragments or derivatives recognize a TAA. The therapeutically
effective immune response which is induced by the vaccination with
suitable antibodies directed against TAA is determined by the
binding region of these antibodies, i.e. by their idiotype.
Therefore, it is, in principle, also possible to use, instead of
intact antibodies, fragments or derivatives of these antibodies for
a successful vaccination as long as these derivatives still contain
the idiotype of the respective starting-antibody. As examples,
without being limiting, can be listed: F(ab)'.sub.2 fragments,
F(ab)' fragments, Fv fragments which can be produced either by
known biochemical methods (enzymatic cleavage) or by known methods
of molecular biology. Further examples are derivatives of
antibodies, which can be produced according to known chemical,
biochemical or gene technological methods. In this context, the
term "derivative", in particular, also includes products which can
be produced by chemical linkage of antibodies (antibody fragments)
with molecules which can enhance the immune response, such as
tetanus toxoid, Pseudomonas exotoxin, derivatives of Lipid A,
GM-CSF, IL-2 or by chemical linkage of antibodies (antibody
fragments) with lipids for a better incorporation into a liposome
formulation. The term "derivative" also includes fusion proteins of
antibodies (antibody fragments), which have been produced gene
technologically, with polypeptides which can enhance the immune
response, such as GM-CSF, IL-2, IL-12, C3d etc. According to the
invention, the antibodies can, of course, also be applied in
combination with each other. This means that two or more antibodies
which recognize different membrane antigens or different epitopes
of the same membrane antigen can be administered. The different
antibodies can be administered simultaneously (together or
separately) or subsequently. Cancer cells often express several TAA
at the same time against which suitable antibodies for vaccination
are either available or can be generated. In order to obtain an
enhanced or possibly synergistic effect of the induced immune
response and to minimize the potential danger of the selection of
antigen-negative variants and in order to counteract a possible
tumor cell heterogenity, it may be advantageous to use a
combination of two or more suitable antibodies or their fragments
or derivatives simultaneously for vaccination.
In the context of the present invention the term "vaccination"
means an active immunization, i.e. an induction of a specific
immune response due to administration (e.g. subcutaneous,
intradermal, intramuscular, possibly also oral, nasal) of small
amounts of an antigen (a substance which is recognized by the
vaccinated individual as foreign and therefore immunogenic) in a
suitable immunogenic formulation. The antigen is thus used as a
"trigger" for the immune system in order to build up a specific
immune response against the antigen. In principle, the required
amounts of the antigen can be very small (some vaccines only
contain about 5-10 .mu.g antigen per dose of vaccination).
It is characteristic of an active immunization that dose-effect
curve depends, over a wide range, only little on the amount of
antigen administered. This means that the immune response is more
or less identical in a wide range of doses. As a consequence, in
the case of vaccination, the desired effect, i.e. the induction of
an immune response, can already be achieved with very small amounts
of antigen. It can, however, also be achieved in a comparable
manner using substantially larger amounts of antigen. It is, of
course, desirable to use, in general, as low a dosage as possible,
in particular in view of side effects, costs for material etc.,
which are of importance as regards vaccination.
In the sense of the present invention a vaccination can, in
principle, be either carried out in the therapeutic sense as well
as in the prophylactic sense (as is the case with all antimicrobial
vaccines). This means that the vaccination against cancer according
to the present invention can be understood as both a therapeutic
and a prophylactic application. Accordingly, it might optionally be
possible to achieve a prophylactic protection against the breakout
of a cancer disease by vaccination of individuals who do not suffer
from cancer. Individuals to whom such a prophylactic vaccination
can be applied are individuals who have an increased risk to
develop a cancer disease, although this application is not limited
to such individuals.
The use according to the present invention differs substantially
from the basic possibilities of therapeutic application of
antibodies for the treatment of cancer that have been known so far
and have been described earlier and allows for an unexpectedly
efficient treatment.
The binding region of an antibody against a TAA can represent a
structural complementary picture of the binding epitope of the
respective TAA according to the "lock and key" principle. This
means that such an antibody has, in its idiotype, a structural
information of the epitope of the TAA against which it is directed.
Thus, if a cancer patient is vaccinated with a suitable immunogenic
antibody against a TAA (i.e. for example with a murine MAB against
a TAA), antibodies are produced in the patient which, in part, are
directed against the idiotype of the antibody used as vaccine and
which can structurally mimic the epitope of the TAA according to
the "lock and key" principle. This means that due to such a
vaccination, so to say, soluble variants of the epitope of the TAA
are generated in the cancer patient, which can be effective as
actively induced autologous antibodies for a long period of time
and the titer of which can be boosted in suitable intervals by
repeated vaccinations.
In a preferred embodiment, the human cellular membrane antigen is a
structure which plays a role in adhesion processes. In this
context, adhesion processes preferably are cell-cell-interactions
wherein ligands or receptors on the cell surface are involved.
Thus, adhesion molecules are ligands or receptors on the cell
surface which serve the function of cell-cell-interaction. A
subgroup of such adhesion molecules are the self-adhesion
molecules. These possess the property to be able to bind to
themselves.
The physiological effect of an immune response induced by
vaccination with an antibody directed against a TAA naturally
depends on the function of the respective TAA. If the TAA has, for
example, the function of a receptor for the adhesion of tumor
cells, in particular to a ligand on endothelial cells of the
vascular system (such a property is important for the ability of
the disseminated tumor cells to exit from the vascular system and
to settle in tissue in order to form a metastasis), this ability
for adhesion is reduced by vaccination with a suitable antibody
directed against this TAA, since induced antibodies, which will
compete for the interaction of the TAA with its ligand as they
mimic the TAA in soluble form, will be permanently present in the
circulation and the tissue.
Generally spoken, it is possible, according to the explanations
given above, to achieve by vaccination with suitable antibodies
against TAA which have a function as regards the malignity of tumor
cells, that the induced immune response interferes with the
function of the TAA in its interaction with its ligand and hampers
or prevents this interaction. This means that cancer cells can not
or not sufficiently express properties which are important for the
malignant phenotype, which makes it possible to slow down or stop
the development of the disease and to suppress the development of
metastases, in particular, at an early stage.
In a further preferred embodiment, the cellular membrane antigen is
capable of self-adhesion, i.e. certain epitopes of the antigen are
responsible for the homophilic binding to the same antigen on
another cell. Examples of such antigens are, inter alia, N-CAM
(Neuronal Cellular Adhesion Molecule), CEA (Carcino Embryonic
Antigen) and Ep-CAM (Epithelial Cell Adhesion Molecule). Antibodies
directed against epitopes of self-adhesion antigens which are
involved in this function, can, as described above, contain a
structural information complementary to such an epitope. By
vaccination with such antibodies, it is thus possible, as described
above, to induce the formation of antibodies which have the
property of this self-adhesion in the binding reaction. This means
that such induced antibodies can, in turn, bind to the
self-adhesion antigen since in such a case receptor and ligand are
identical. Thus, it is possible to induce an immune response by
vaccination of cancer patients with suitable antibodies directed
against self-adhesion antigens, wherein said immune response in
turn directly binds to tumor cells and thereby triggers various
therapeutic effects. On the one hand, the ability of self-adhesion,
which is important to malignant cells, is blocked and, on the other
hand, human effector functions such as complement-dependent lysis
and/or lysis due to activation of cytotoxic effector cells, can be
triggered by the binding of the induced antibodies to the tumor
cells, which lead to the destruction of the tumor cells.
By all the above mentioned mechanisms and effects, the formation of
new metastases can be suppressed and the dissemination of the
disease can, at least, be slowed down thanks to vaccination of
cancer patients with suitable antibodies against TAA. In early
stages of the disease, for example after a successful operation of
a primary tumor (adjuvant stage), remaining disseminated tumor
cells are prevented from establishing themselves as new metastases
due to such vaccinations. This allows to prolong the relapse-free
survival period and therefore the overall lifetime of such
patients. It may optionally be possible to obtain a lifelong
protection against the formation of metastases due to such
vaccinations and booster vaccinations which are carried out in
suitable intervals. Of particular interest are vaccinations of
cancer patients with suitable antibodies directed against a
self-adhesion TAA since in these cases, as described above, it is
possible to achieve an enhanced therapeutic effect due to an
additional direct attack of the induced immune response on the
tumor cells.
In a further preferred embodiment, the pharmaceutical composition
prepared according to the use of the present invention contains at
least one adjuvant commonly used in the formulation of vaccines
apart from the antibody. It is possible to enhance the immune
response by such adjuvants. As examples of adjuvants, however not
being limited to these, the following can be listed: aluminium
hydroxide (Alu gel), derivatives of lipopolysaccharides, Bacillus
Calmette Guerin (BCG), liposome preparations, formulations with
additional antigens against which the immune system has already
produced a strong immune response, such as for example tetanus
toxoid, Pseudomonas exotoxin, or constituents of influenza viruses,
optionally in a liposome preparation, biological adjuvants such as
Granulocyte Macrophage Stimulating Factor (GM-CSF), interleukin 2
(IL-2) or gamma interferon (IFN.gamma.).
In another preferred embodiment, the pharmaceutical composition
prepared according to the use of the invention is suitable for
administration for vaccination in a dosage of 0.01 to 4 mg
antibody, preferably of 0.5 mg.
The vaccination can be carried out by a single application of the
above mentioned dosage. However, preferably the vaccination is
carried out by repeated applications. The number of repetitions is
in the range from 1 to 12 per year, more preferably in the range
from 4 to 8 per year. The dosage can remain the same or can
decrease.
Booster vaccinations can be carried out in regular intervals, in
principle, lifelong. Suitable intervals are in the range from 6 to
24 months and can be determined by monitoring the titer of the
induced antibodies (a booster vaccination should be carried out as
soon as the titer of the induced antibodies has dropped
significantly).
The administration of the antibody can be carried out according to
methods known to the person skilled in the art. Preferably, the
pharmaceutical composition containing the antibody is suitable for
a subcutaneous, intradermal or intramuscular administration.
The present invention furthermore relates to the use of antibodies
which recognize a tumor-associated antigen for the vaccination
against cancer diseases as well as to a method for treating cancer
diseases by vaccination, wherein one or more antibodies which
recognize a TAA are administered to a patient in an amount
sufficient for vaccination. For the definitions and the preferred
embodiments the same holds true as already described above in
connection with the use according to the invention.
The use of antibodies directed against TAA or of their derivatives
or fragments as vaccines differs substantially from the known
applications of such anti-TAA antibodies for the passive
immunotherapy. Some essential advantages of the use according to
the invention in comparison to the passive antibody immuno therapy
are summarized as follows:
Antibodies Directed Against TAA for the Passive Immunotherapy of
Cancer:
high dosage (>100 mg/intravenous infusion) short effect due to
elimination of the effective agent xenogenic antibody undesirable
due to immunogenity the duration of the therapy is limited, in
particular in the case of xenogenic antibodies, due to the
induction of an immune response and the danger of anaphylactic
reactions caused thereby in the case of repeated applications
Antibodies Directed Against TAA for the Prophylactic and/or
Therapeutic Vaccination Against Cancer: low dosage (<1
mg/vaccination; subcutaneous, intradermal or intramuscular
injection) long lasting effect of the directly induced immune
response xenogenic antibodies desirable since the effect is based
on immunogenicity duration of the treatment unlimited (booster
vaccinations are always possible)
In the following, experiments will be described which show that the
vaccination with a certain murine MAB (HE2), which is directed
against the self-adhesion TAA Ep-CAM, or the vaccination with its
F(ab)'.sub.2 fragment directly leads to the induction of antibodies
which selectively bind on human tumor cells expressing this
antigen. This shows, as an example but without any limitation, that
an immune response which can have a therapeutic effect in cancer
diseases is induced by vaccination with suitable antibodies
directed against a self-adhesion TAA or with their derivatives
which, at least, comprise the idiotype of the starting
antibody.
For this purpose, the murine monoclonal antibody HE2 was generated
according to described standard procedures of the hybridoma
technology (see, e.g., H. Zola. Monoclonal Antibodies: A Manual of
Techniques. CRC Press, Inc. ISBN 0-8493-6476-0; 1988). Balb/c mice
were immunized with human colorectal cancer cells according to
standard protocols. The spleen cells were fused with the mouse
melanoma line P3X63Ag8 and hybridomas were selected which produce
antibodies which selectively bind to human colorectal cancer cells
but not to melanoma cells. Finally, a hybridoma was selected which
secreted an IgG2a/kappa antibody. This antibody (HE2) binds to
Ep-CAM as can be shown, e.g., by Western Blot analysis with
membrane preparations from KATO IlIl stomach cancer cells using a
known anti-Ep-CAM antibody (KS1-4) as a comparison.
The amino acid sequences of the variable regions of MAB HE2 are as
follows:
TABLE-US-00001 Heavy chain: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEW
(SEQ ID NO: 1) VKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTA
DKSSSTAYMQLSSLTSDDSAVYFCARDGPWFAYWGQ GTLVTVSA Light chain:
NIVMTQSPKSMSMSVGERVTLTCKASENVVTYVSWY (SEQ ID NO. 2)
QQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFT
LTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIK
The figures show:
FIG. 1 shows the inhibition of the self-adhesion of the small cell
lung cancer line SW2 by the MAB HE2 in vitro.
FIG. 2 shows the self-adhesion of the human small cell lung cancer
line SW2 without the influence of the MAB HE2 in vitro as a control
to the experiment shown in FIG. 1.
FIG. 3 shows the induction of an antibody immune response against
HE2 after vaccination of goats with the F(ab)'2 fragment of HE2 as
determined in an ELISA.
FIG. 4 shows the induction of an antibody immune response against
Ep-CAM positive human stomach cancer cells (Kato III) after
vaccination of goats with the F(ab)'2 fragment of HE2 as determined
in a cell-ELISA.
FIG. 5 shows the absence of an antibody immune response against
Ep-CAM negative human melanoma cells (WM9) after vaccination of
goats with the F(ab)'2 fragment of HE2 as determined in a
cell-ELISA, which was carried out as a control to the experiment
shown in FIG. 4.
FIG. 6 shows the binding of an affinity purified antibody fraction
from serum of goats, which were vaccinated with the F(ab)'2
fragment of HE2, to Ep-CAM positive human stomach cancer cells
(Kato III) as determined in a cell-ELISA.
FIG. 7 shows the absence of the binding of an affinity purified
antibody fraction from serum of goats, which were vaccinated with
the F(ab)'2 fragment of HE2, to Ep-CAM negative human melanoma
cells (WM9) as determined in a cell-ELISA, which was carried out as
control to the experiment shown in FIG. 6.
FIG. 8 shows the induction of an antibody immune response against
HE2 after vaccination of rhesus monkeys with 0.5 mg HE2 adsorbed to
aluminium hydroxide as determined in an ELISA.
FIG. 9 shows the induction of an antibody immune response against
Ep-CAM positive human stomach cancer cells (Kato III) after
vaccination of rhesus monkeys with 0.5 mg HE2 adsorbed to aluminium
hydroxide as determined in a cell-ELISA.
FIG. 10 shows the induction of an antibody immune response against
HE2 detected in connection with a toxicity study with rhesus
monkeys after vaccination of one group of rhesus monkeys with 0.5
mg HE2 adsorbed to aluminium hydroxide as well as the absence of an
immune response against HE2 after treatment of another group of
rhesus monkeys with an aluminium hydroxide formulation without
antigen (placebo) as determined in an ELISA.
FIG. 11 shows exemplarily the induction of an antibody immune
response against Ep-CAM positive human stomach cancer cells (Kato
III) detected in connection with the toxicity study of rhesus
monkeys with HE2 as determined in a cell-ELISA.
FIG. 12 shows the induction of an antibody immune response against
Ep-CAM positive human stomach cancer cells (Kato III) after
repeated vaccination of a patient suffering from intestinal cancer
with 0.5 mg HE2 adsorbed to aluminium hydroxide, as determined in a
cell-ELISA.
FIG. 13 shows the induction of a serum cytotoxicity against Ep-CAM
positive human stomach cancer cells (Kato III) after repeated
vaccination of a patient suffering from intestinal cancer with 0.5
mg HE2 adsorbed to aluminium hydroxide, as determined in a cell
lysis experiment.
The following examples serve to further illustrate the invention
but shall not limit it:
In order to show that the murine MAB HE2 binds to an epitope of the
self-adhesion antigen Ep-CAM, which is involved in the homophilic
binding, the influence of HE2 on the ability for self-adhesion of
the human cell line SW2 was investigated. This small cell lung
carcinoma line tends to form cell clusters in in vitro culture
within several hours after preceding single seeding. The
description of the experiment can be found in Example 1. As is
evident from FIGS. 1 and 2, the formation of cell clusters is
prevented to a large extend by the addition of HE2. This proves
that HE2 binds to an epitope of Ep-CAM which is involved in the
homophilic binding of this membrane protein.
In order to be able to investigate the direct humoral immune
response to the vaccination with the F(ab)'.sub.2 fragment of the
murine MAB HE2, goats were immunized with this fragment. The
fragment was prepared according to methods that are known and
described by cleavage of HE2 with pepsin and was purified. The
immunization of the goats is described in Example 2.
First, the goat immunoserum that was recovered and pooled was
investigated, in comparison to a pre-serum, for immuno globulins
which are directed against the MAB HE2 in order to determine the
total immune response of the vaccinated goats. This investigation
was carried out with the help of an ELISA assay, the experimental
description of which is given in Example 3. The result of this
experiment is shown in FIG. 3: the goats have, due to the
vaccination with the F(ab)'.sub.2 fragment of the MAB HE2,
developed a strong immune response thereto, whereas no antibodies
against HE2 could be found in the pre-serum.
In the following, it was investigated whether it is possible to
detect immunoglobulins in the goat immunoserum, which bind to human
cancer cells which express the TAA against which the MAB HE2 is
directed (Ep-CAM). For this purpose, the stomach cancer cell line
KATO III was used. Also the binding to a human cell line, which
does not express Ep-CAM (WM9 melanoma cells), was tested as a
control. These investigations were carried out with the help of
cell-ELISA assays, the experimental description of which is given
in Example 4. The results of these experiments are shown in FIGS. 4
and 5: the goat immunoserum contains immunoglobulins which strongly
bind to the Ep-CAM positive KATO cells, whereas no binding can be
detected on the Ep-CAM negative WM9 cells. The pre-serum contains
no antibodies which bind to these cells. This very surprising
result shows that antibodies generated by the vaccination with the
HE2-F(ab)'.sub.2 fragment are indeed capable to bind themselves
again to cells which express the TAA recognized by HE2.
Consequently, the function of the TAA of self-adhesion could be
transferred to the antibodies which were generated by the
vaccination with HE2, as previously described in detail.
In order to prove that the antibodies produced in the goats due to
the vaccination with the F(ab)'.sub.2 fragment of HE2 and which are
directed against the idiotype of this MAB are indeed those which
bind to the KATO cells, the anti-idiotypic portion of these induced
antibodies was specifically purified from the goat immunoserum with
the help of a sequence of immunoaffinity chromatographies as
principally described (Proc. Natl. Acad. Sci. USA 81 (1984), 216).
The sequence of the purification steps is again summarized in
Example 5.
These affinity purified goat antibodies were again tested for their
binding to the Ep-CAM positive KATO cells as well as to the Ep-CAM
negative WM9 cells. The experimental description is given in
Example 6. The result of these experiments is shown in the FIGS. 6
and 7: the goat IgG, which is directed against the idiotype of HE2,
binds strongly to the Ep-CAM positive KATO cells, whereas
unspecific goat IgG hardly binds. The binding of the affinity
purified specific goat IgG to the Ep-CAM negative WM9 cells,
however, does not differ from that of the unspecific goat IgG. It
is thus proven that the fraction of the antibodies which directly
developed due to the vaccination with the F(ab)'.sub.2 fragment of
HE2 and which are directed against the idiotype of this antibody,
contains the antibodies which bind to the cancer cells which
express the TAA recognized by HE2. By this experiment it is also
conclusively shown that the antibodies against Ep-CAM positive
cells induced by the vaccination with HE2 are not the result of a
double autologous idiotypic network cascade as was postulated in
several publications (see, e.g.: Cancer Immunol. Immunother. 42
(1996), 81-87), for such anti-idiotypic antibodies (Ab3) could not
at all be purified by affinity chromatography on an Ab1 (=HE2)
column since, according to the idiotypic network, they cannot bind
to Ab1 but only to Ab2.
In view of the above described results of the immunization of goats
with the F(ab)'.sub.2 fragment of HE2, vaccination studies were
also carried out with rhesus monkeys in order to confirm the
immunological results in a species closely related to human. For
these experiments, the complete murine MAB HE2 was used as
immunogen. It was assumed that the murine Fc part as a large
xenogeneic protein would also enhance the immune response against
the idiotype (carrier effect). In order to avoid possible local
side effects, aluminium hydroxide was used as a mild adjuvant. The
preparation of the formulation for these vaccination experiments is
described in Example 7.
The formulation described in Example 7 was injected subcutaneously
in the back of four rhesus monkeys (0.5 mg HE2=0.5 ml per
vaccination, administered two times at an interval of four weeks).
For the recovery of serum, blood was taken at several points of
time.
First, the immune response against HE2 was determined in an ELISA.
The experimental description is given in Example 8. As shown in
FIG. 8, significant titers of antibodies against HE2 can already be
measured on the day 29.
It was furthermore investigated whether antibodies are induced by
the vaccination which bind to KATO III cells. For these tests, a
cell-ELISA was used. The experimental description is given in
Example 9. As shown in FIG. 9, antibodies which bind to Ep-CAM
positive Kato III tumor cells are already induced on day 29 in all
animals.
In the following, four animals were vaccinated with HE2 adsorbed to
aluminium hydroxide in connection with a toxicity study with rhesus
monkeys. Four other rhesus monkeys received aluminium hydroxide as
a placebo. The preparation of the formulations is described in
Examples 10 and 11. In total, the rhesus monkeys were injected
subcutaneously in the back four times with 0.5 ml of the respective
formulation (effective agent or placebo) (days 1, 15, 29 and 57).
For the recovery of serum, blood was taken before the start of the
study and at different times during the treatment.
Again, the immune response against HE2 was first determined in an
ELISA. The experimental description is given in Example 8. As shown
in FIG. 10, all four rhesus monkeys of the HE2 group developed a
significant humoral immune response against HE2 already after one
vaccination which was further enhanced by the second vaccination,
whereas the rhesus monkeys of the placebo group do not show any
increase in the titer of antibodies against HE2.
These findings were further confirmed by immunoaffinity
purification of the sera of day 43 of the monkeys of the HE2 group.
The experimental description is given in Example 12. As shown in
the following table, all four monkeys have developed a strong IgG
immune response against HE2 (secondary immune response) in their
serum on day 43, whereas the IgM portion is comparable to that of
the pre-sera.
TABLE-US-00002 monkey day .mu.g/ml IgM against .mu.g/ml IgG against
9206m -14 7.7 2.8 43 16.3 135.2 9599m -14 17.9 2.5 43 25.4 449.3
8415f -14 16.0 3.2 43 22.5 159.9 9139f -14 5.3 5.0 43 10.3 69.8
Also the induction of antibodies against Ep-CAM positive Kato III
cells was investigated. Again, a cell-ELISA was used for these
tests. The experimental description is given Example 9. As is shown
exemplarily in FIG. 11, rhesus monkeys of the HE2 group developed
antibodies against Kato III cells already on day 29.
In view of the above described results of the vaccination of goats
and rhesus monkeys, a patient suffering from intestinal cancer with
metastases (Dukes D) was in the following vaccinated with the MAB
HE2, adsorbed to aluminium hydroxide, in an anecdotal case. The
preparation of the formulation is described in Example 7. In total,
the patient was injected four times (day 1, 50, 78, 114)
subcutaneously in the upper extramities with 0.5 ml of this
formulation (corresponds to 0.5 mg HE2). Blood was taken for the
recovery of serum prior to each vaccination and on day 128. First,
it was investigated whether antibodies were induced by the
vaccination which bind to KATO III cells. The cell-ELISA was again
used for these tests. The experimental description is given in
Example 9. The results of these experiments are shown in FIG. 12
High titers of antibodies which bind to KATO III cells are
obviously induced in this cancer patient due to the
vaccination.
It was furthermore investigated whether the antibodies induced by
the vaccination with HE2 mediate a cytotoxic effect against KATO
III cancer cells ex vivo. For this purpose, KATO III cells were
incubated with pre- and immunosera of this cancer patient in order
to demonstrate a complement-dependent lysis mediated by the induced
antibodies. The experimental description is given in Example
13.
The results are shown in FIG. 13. The antibodies induced by the
vaccination with HE2 are obviously able to destroy Ep-CAM positive
KATO III cells via complement-dependent lysis in autologous patient
serum.
The above described experiments exemplarily show that the
vaccination with suitable antibodies against a self-adhesion TAA,
such as Ep-CAM, or their derivatives with the same idiotype as the
respective starting antibodies, triggers a humoral immune response
which selectively binds on tumor cells which express this
self-adhesion TAA. The induced antibodies may display a cytotoxic
potential against such tumor cells. A vaccination with such
antibodies can therefore lead to a therapeutic effect in cancer
diseases.
EXAMPLES
TABLE-US-00003 Materials used: microtiter plates: Immuno Plate F96
MaxiSorp (Nunc) for ELISA Cell Culture Cluster (Costar; Cat.Nr.
3598) for cell-ELISA cell lines: SW2: human small cell lung
carcinoma line, Ep-CAM positive KATO III: human stomach cancer cell
line, Ep-CAM positive (ATCC HTB 103) WM 9: human melanoma cell
line, Ep-CAM negative Coupling buffer: 0.1 M NaHCO.sub.3 0.5 M NaCl
pH value 8.0 Purification buffer A: PBS def 0.2 M NaCl pH value 7.2
Purification buffer B: 0.1 M glycine/HCl 0.2 M NaCl pH value 2.9
Medium A: RPMI 1640 + 2 g/l NaHCO.sub.3 100 U/ml penicillin G 100
.mu.g/ml streptomycin sulfate 4 mM glutamine 10% fetal calf serum
(heat inactivated) Binding buffer: 15 mM Na.sub.2CO.sub.3 35 mM
NaHCO.sub.3 3 mM NaN.sub.3 pH value: 9.6 PBS deficient: 138 mM NaCl
1.5 mM KH.sub.2PO.sub.4 2.7 mM KCl 6.5 mM Na.sub.2HPO.sub.4 pH
value: 7.2 Fixing solution: 0.1% glutardialdehyde in physiological
NaCl solution Washing buffer A: 2% NaCl 0.2% Triton X-100 in PBS
deficient Washing buffer B: 0.05% Tween 20 in PBS deficient
Blocking buffer A: 5% fetal calf serum (heat inactivated) in PBS
deficient Blocking buffer B: 1% bovine serum albumin 0.1% NaN.sub.3
in PBS deficient Dilution buffer A: 2% fetal calf serum (heat
inactivated) in PBS deficient Dilution buffer B: PBS deficient
Staining buffer: 24.3 mM citric acid 51.4 mM Na.sub.2HPO.sub.4 pH
value: 5.0 Substrate: 40 mg o-phenylen diamin dihydrochloride 100
ml staining buffer 20 .mu.l H.sub.2O.sub.2 (30%) Stop solution: 4 N
H.sub.2SO.sub.4
Example 1
In vitro cultivated SW2 cells are centrifuged and the pellet is
suspended in Medium A and adjusted to 7.times.10.sup.4 cells/ml. In
the chambers of a LabTek either 0.1 ml PBS def are mixed with 0.3
ml of the cell suspension or 0.1 ml PBS def are mixed with 40 .mu.g
HE2 and then with 0.3 ml of the cell suspension (final
concentration of HE2 100 .mu.g/ml). Just before the cell suspension
is added as the last constituent, the cells are separated with the
pipette. Immediately after mixing, the respective cell suspensions
are photographed in the inverted microscope (magnification
100-fold). Subsequently, the cell suspensions are cultivated for 4
hours at 37.degree. C./5% CO.sub.2 and then photographed again.
Example 2
Two goats are each vaccinated intradermally at multiple sites with
1.5 mg of the F(ab)'.sub.2 fragment in 3 ml PBS deficient together
with 3 ml of Freund's Complete Adjuvant (Difco). On day 8, a first
booster vaccination as on day 1 is given, however with Freund's
Incomplete Adjuvant (Difco). On day 29, a second booster
vaccination is given in the same manner. However, no adjuvant is
added. Blood is taken before the start of the vaccination and on
day 54 for the recovery of serum for the analysis of the immune
response developed.
Example 3
100 .mu.l aliquots of the MAB HE2 (solution with 10 .mu.g/ml in
binding buffer) are incubated in the wells of a microtiter plate
for 1 hour at 37.degree. C. After washing the plate with washing
buffer A six times, 200 .mu.l of the blocking buffer A are added to
each well and the plate is incubated for 30 minutes at 37.degree.
C. After washing the plate as described above, 100 .mu.l aliquots
of the goat sera to be tested are incubated in dilutions from 1:100
to 1:1 000 000 in dilution buffer A for 1 hour at 37.degree. C.
After washing the plate as described above, 100 .mu.l of the
peroxidase-conjugated rabbit anti-goat-Ig antibody (Zymed) are
added to each well at a dilution of 1:1000 in dilution buffer A and
are incubated for 30 minutes at 37.degree. C. The plate is washed
with washing buffer A for four times and twice with staining
buffer. The binding of the antibody is detected by addition of 100
.mu.l of the specific substrate to each well and the staining
reaction is stopped after about 10 minutes by addition of 50 .mu.l
stop solution. The evaluation is carried out by measuring the
optical density (OD) at 490 nm (wavelength of the reference
measurement is 620 nm).
Example 4
The wells of a microtiter plate were incubated at +4.degree. C.
over night with 100 .mu.l of a cell suspension of the cell line to
be tested at a concentration of 2.times.10.sup.6 cells/ml in medium
A. After sucking off the supernatant, the plate is incubated with
50 .mu.l fixing solution per well for 5 minutes at room
temperature. After sucking off the supernatant, 200 .mu.l blocking
buffer B are added to each well and the plate is incubated for 1
hour at 37.degree. C. After washing twice with 200 .mu.washing
buffer B, 100 .mu.l aliquots of the goat sera to be tested are
incubated for 1 hour at 37.degree. C. at dilutions of 1:10 to 1:100
000 in dilution buffer B. After washing the plate twice with 100
.mu.l ice-cold washing buffer B, 100 .mu.l of the
peroxidase-conjugated rabbit anti-goat-Ig antibody (Zymed) are
added at a dilution of 1:1000 in dilution buffer A and are
incubated for 45 minutes at 37.degree. C. The plate is washed three
times with 100 .mu.l ice-cold washing buffer B. The binding of the
antibody is detected by the addition of 100 .mu.l of the specific
substrate per well and the staining reaction is stopped after about
10 minutes by addition of 50 .mu.l stop solution. The evaluation is
carried out by measuring the optical density (OD) at 490 nm
(wavelength of the reference measurement is 620 nm).
Example 5
The purification is principally described in Proc. Natl. Acad. Sci.
USA 81:216, 1984 and is summarized as follows: in a first step, a
purification of the total IgG contained in the goat serum is
carried out according to known methods on a DEAE anion exchanger
column. Subsequently, the goat antibodies which are directed
against constant regions of the F(ab)'.sub.2 fragment of HE2 are
bound to an immunoaffinity column (CH-Sepharose 4B, Pharmacia) to
which irrelevant murine IgG2a was coupled, whereas the fraction of
the anti-idiotypic goat antibodies does not bind to this column.
Therefore, in a last step, the flow-through of this immunoaffinity
chromatography is bound to an immunoaffinity column (CH-Sepharose
4B, Pharmacia) to which HE2 was coupled. The fraction specifically
bound to this column is eluted with a buffer pH 2.8 (0.1 M
glycine/HCI) and neutralized. The goat IgG fraction obtained in
this way is directed against the idiotype of HE2.
Example 6
This cell-ELISA is basically carried out in the same way as
described in Example 4. Instead of serum dilutions, concentrations
of 100 .mu.g/ml to 0.031 .mu.g/ml of the immunoaffinity-purified
goat IgG and of the unspecific purified goat IgG, respectively, are
used.
Example 7
0.83 ml of a suspension of Alu-Gel (Alu-Gel S by Serva, 2%
suspension, quality degree: adjuvant for the preparation of
vaccines) is carefully agitated for 1 hour at room temperature
under sterile conditions with 0.5 ml of a solution of 10 mg/ml HE2
in PBS pH 5.5 together with 3.67 ml PBS def. (final concentration
of HE2: 1 mg/ml; Alu-Gel S: 0.33%). Then, the suspension is
sterilly filled in injection vials at aliquots of 0.5 ml.
Example 8
This ELISA is basically carried out in the same manner as described
in Example 3 with the exception that a peroxidase-conjugated
goat-anti-human-Ig antibody (Zymed) is used for the detection of
the bound rhesus monkey antibodies. With this reagent rhesus monkey
antibodies can be detected in the same manner as human antibodies
since the sequence homology of the constant regions of human
antibodies and rhesus monkey antibodies is about 98%.
Example 9
This cell-ELISA is basically carried out in the same manner as
described in Example 4 with the exception that a
peroxidase-conjugated goat-anti-human-lg antibody (Zymed) is used
for the detection of the rhesus monkey antibodies (or the human
antibodies) which are bound to the cells. A peroxidase-conjugated
goat-anti-mouse-lgG antibody (Zymed) is used for the detection of
the murine HE2 as a control.
Example 10
3.5 ml of a solution of HE2 (10 mg/ml in PBS def. pH=5.5) are mixed
under sterile conditions with 0.35 ml of an aqueous thimerosal
solution (10 mg/ml; Sigma) as well as with 27.25 ml physiological
saline solution and added to 3.9 ml of an aluminium hydroxide
suspension (3% in water; Alhydrogel, Superfos Biosector, Denmark)
under careful agitation. 0.6 ml of the suspension obtained in this
way are then filled into depyrogenated glass tubes under sterile
conditions which are sealed with a rubber plug with an aluminium
cap.
Example 11
The placebo formulation is prepared in the same manner as described
in Example 10 with the exception that 0.35 ml physiological NaCl
solution is used instead of the antibody solution and 3.5 ml PBS
def pH=5.5 and instead of the thimerosal solution.
Example 12
1 g CH-Sepharose 4B (Parmacia) are suspended in 30 ml 1 mM HCl for
15 minutes. The gel is then washed on a filter of sintered glass
AG3 with 1 liter 1 mM HCl and subsequently with 200 ml coupling
buffer. 10 mg HE2 (stock solution 10 mg/ml) are dialyzed against
about 0.5 liter coupling buffer. This solution is mixed with the
gel suspension in a sealed container. A ratio of gel: buffer of 1:2
leads to a suspension suitable for the coupling. This suspension is
agitated for 5.5 hours at room temperature. Subsequently, the
excess of the ligand is removed by washing with 3.times.30 ml
coupling buffer. Remaining reactive groups are blocked by a 1 hour
incubation at room temperature with 1 M ethanol amine. The gel is
then agitated for 1 hour at room temperature with 0.1 M Tris-HCl
buffer pH=8. Finally, the gel is washed with 3 cycles of buffers
with alternating pH. Each cycle consists of 0.1 M sodium acetate
buffer pH 4 with 0.5 M NaCl, and subsequently 0.1 M Tris-HCl buffer
pH 8 with 0.5 M NaCl. The gel is kept at 4.degree. C.
The immunoaffinity purification of the antibody fraction directed
against HE2 from the serum of the rhesus monkeys is carried out
according to the following instructions: the immunoaffinity
purification is carried out on the FPLC system (Pharmacia). 1 ml of
the gel obtained according to the above instructions is filled into
a Pharmacia HR5/5 column. 0.5 ml serum are diluted 1:10 with
Purification buffer A. This solution is pumped over the column at a
rate of 1 ml/minute and washed with purification buffer A until the
UV basis line of the detector is reached again (280 nm). Bound
immunoglobulines are then eluted with Purification buffer B and the
fraction is immediately neutralized after desorption with 0.5 M
Na.sub.2HPO.sub.4 and 0.02% NaN.sub.3 are added. 50 .mu.l of the
antibody fraction purified in this way are analyzed on a size
fractionation column (SEC, Zorbax 250 GF) and the portions of IgG
and IgM are quantified. For the SEC 220 mM phosphate buffer pH
7+10% acetonitrile is used as an eluent. Human IgG and human IgM
serve as standard for the SEC which were each chromatographed in
several concentrations for establishing a standard calibration
curve (peak area vs. concentration). The calculation of the IgG and
IgM concentrations in the affinity purified antibody fractions from
rhesus monkeys was carried out by linear regression using the
standard curves. The concentrations are indicated as pg/ml of the
used monkey serum.
Example 13
One day before carrying out the test, KATO III cells are
transferred to fresh medium A and are kept at 37.degree. C./5%
CO.sub.2 in a cell culture flask. On the next day, the cells are
first labelled with .sup.51chrome. 5.times.10.sup.6 cells are
incubated in 800 .mu.l medium A at 37.degree. C./5% CO.sub.2 with
100 .mu.Ci Na.sub.2.sup.51CrO.sub.4. Subsequently, the cells are
washed with medium A and adjusted to a density of
2.5.times.10.sup.5 cells/ml. 100 .mu.l aliquots of this cell
suspension are pipetted into the wells of a microtiter plate. 100
.mu.l aliquots of the patient sera to be tested are added and
incubated for 3 hours at 37.degree. C./5% CO.sub.02 (the sera are
stored at--80.degree. C. and are thawed only once for this assay in
order to avoid harming the activity of the complement). The
supernatants are recovered by using a Skatron-Harvesting-Press and
are measured in a gamma-counter. As a result, the values for the
"experimental release" are obtained. For the determination of the
"total release", the cells are treated as described above wherein
serum is replaced by a solution of 2% SDS, 50 mM Na.sub.2CO.sub.3
and 10 mM EDTA. The values for the "spontaneous release" are
obtained by replacing serum by medium A. The result is calculated
as follows:
.times..times..times..times..times..times..times..times..times..times..ti-
mes. ##EQU00001##
The test is carried out 3 times and the mean value and the standard
deviation of the single results are indicated.
SEQUENCE LISTINGS
1
21116PRTMus musculus 1Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Arg Pro Gly Thr 1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ala Phe Thr Asn Tyr 20 25 30Leu Ile Glu Trp Val Lys Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Asn Pro Gly Ser
Gly Gly Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu
Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80Met Gln Leu Ser
Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Asp
Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ala 1152107PRTMus musculus 2Asn Ile Val Met Thr Gln Ser Pro
Lys Ser Met Ser Met Ser Val Gly 1 5 10 15Glu Arg Val Thr Leu Thr
Cys Lys Ala Ser Glu Asn Val Val Thr Tyr 20 25 30Val Ser Trp Tyr Gln
Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser
Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Ser
Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80Glu
Asp Leu Ala Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr 85 90
95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
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